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| United States Patent |
5,223,519
|
|
Hirano
, et al.
|
June 29, 1993
|
4-hydroxypyridine derivatives, useful for treating circulatory diseases
Abstract
4-Hydroxypyridine derivative represented by the following formula
##STR1##
wherein R represents a methyl group or an isopropyl group, and the method
for preparing the derivative comprising the step of cultivating a strain
belonging to Streptomyces karnatakensis are disclosed. Also disclosed are
the pharmaceutical composition comprising the same and a method for
treating circulatory diseases comprising the step of administering said
compound to a patient.
| Inventors:
|
Hirano; Shin-ichi (Chigasaki, JP);
Watanabe; Yoshio (Fujisawa, JP);
Kominato; Kaichiro (Yamato, JP);
Agata; Naoki (Fujisawa, JP);
Hara; Yutaka (Yokohama, JP);
Shibamoto; Norio (Chigasaki, JP);
Yoshioka; Takeo (Ayase, JP);
Kioka; Tomoyuki (Yokohama, JP);
Iguchi; Hiroshi (Yokohama, JP);
Shirai; Masataka (Tokyo, JP);
Tone; Hiroshi (Yokohama, JP);
Okamoto; Rokuro (Fujisawa, JP)
|
| Assignee:
|
Mercian Corporation (Tokyo, JP)
|
| Appl. No.:
|
830202 |
| Filed:
|
December 13, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
514/348; 435/253.5; 546/296 |
| Intern'l Class: |
A61K 031/44; C07D 213/69; C12N 001/20 |
| Field of Search: |
546/296
514/348
|
References Cited
Other References
Yano, et al., "Actinopyrones A,B and C, new physiologically active
substances. II. Physicochemical properties and chemical structures",
Chemical Abstracts, vol. 105, No. 25, Dec. 1986, p. 747, col. 1, 226123r.
Jansen, et al., "Revised stereochemistry for pipericidin A.sub.1 ",
Chemical Abstracts, vol. 100, No. 15, Apr. 1984, p. 561, col. 2, 120743z.
Yoshida, et al., "The structural revision of pipericiden A by combination
of CMR spectroscopic and biosynthetic studies", Chemical Abstracts, vol.
87, No. 5, Aug. 1977, p. 497, col. 1, 39240z.
|
Primary Examiner: Rotman; Alan L.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Claims
What is claimed is:
1. A 4-hydroxypyridine derivative represented by the following formula (I):
##STR7##
where in R represents a methyl group or an isopropyl group.
2. A method for treating circulatory diseases which comprises the step of
administering to a mammal in need of said treatment an effective amount of
4-hydroxypyridine derivative represented by the following formula (I):
##STR8##
wherein R represents a methyl group or an isopropyl group.
3. The method according to claim 2 wherein the mammal is a human being.
4. The method according to claim 2 wherein the disease is hypertension.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel 4-hydroxypyridine derivative
useful as a vasodilator and vasodepressor, a method for preparing the
4-hydroxypyridine derivative, and a pharmaceutical composition comprising
the derivative.
More specifically, the present invention relates to a 4-hydroxypyridine
derivative represented by the following formula (I):
##STR2##
wherein R represents a methyl group or an isopropyl group. The present
invention also relates to a method for preparing the 4-hydroxypyridine
derivative comprising the step of cultivating a microorganism, a
pharmaceutical composition comprising an effective amount of the
derivative, and a method for treating circulatory diseases comprising the
step of administering the derivative.
2. Description of the Related Art
Yoshida et al. (Argic. Biol. Chem., 41: 849-862, 1977) discloses a series
of Piericidins represented by the following formulas:
##STR3##
wherein R.sub.1 represents a hydrogen atom or a methyl group, and
R.sub.2 represents a methyl group or an isopropyl group.
Yano et al. (J. Antibiotics 39: 38-43, 1986) discloses series of
Actinopyrones represented by the following formula:
##STR4##
The physicochemical properties of Piericidins and Actinopyrones are similar
to those of the 4-hydroxypyridine compounds of the present invention
represented by the above-described formula (I). However, the
4-hydroxypyridine compounds of the present invention structually differ
from Piericidins, because each Piericidine has two or three methoxyl
substituents in its molecule, while both of the compounds of the present
invention have only one methoxyl group in thir molecules. Actinopyrones
are also not structural analogues of the 4-hydroxypyridine compounds of
the present invention, because Actinopyrone is characterized as having a
pyrone nucleous, while the compounds of the present invention have a
pyridine ring structure.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel 4-hydroxypyridine
compounds which can be useful as vasodilators and vasodepressing agents.
Another object of the present invention is to provide a method for
preparing the 4-hydroxypyridine compounds.
Further objects of the present invention are to provide a pharmaceutical
composition comprising an effective amount of the 4-hydroxypyridine
compound, and a method for treating circulatory diseases comprising the
step of administering the 4-hydroxypyridine compound or the pharmaceutical
composition.
Vasodilating activity can be induced by a variety of physiological and
biological factors, and thus, various types of compounds have been
screened for vasodilating activity. The inventors of the present invention
have conducted extensive screening to achieve the foregoing objects and
found that the objects can be effectively attained by providing novel
4-hydroxypyridine compound represented by the above-described formula (I).
The inventors have also found that the compounds of the present invention
having vasodilating activity and vasodepressor activity are useful for the
treatment of circulatory diseases.
Thus, in accordance with the above objects, the present invention provides
a novel 4-hydroxypyridine derivative represented by the following formula:
##STR5##
wherein R represents a methyl group or an isopropyl group.
In accordance with another embodiment of the present invention, there is
provided a method for preparing the novel 4-hydroxypyridine derivatives
which comprises the steps of cultivating a strain belonging to
Streptomyces karnatakensis which can produce said compound.
In accordance with further embodiments, the present invention provides a
pharmaceutical composition comprising an effective amount of the
4-hydroxypyridine derivative, and a method for treating circulatory
diseases which comprises the step of administering to a patient an
effective amount of said 4-hydroxypyridine derivative.
Further objects, features and advantages of the present invention will
become apparent from the Description of the Preferred Embodiments which
follows, when read in light of the accompanying Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. 1 shows a 400 MHz .sup.1 H NMR spectrum of the compound of the present
invention wherein R is a isopropyl group;
FIG. 2 shows a 100 MHz .sup.13 C NMR spectrum of the compound of the
present invention wherein R is a isopropyl group;
FIG. 3 shows a 400 MHz .sup.1 H NMR spectrum of the compound of the present
invention wherein R is a methyl group; and
FIG. 4 shows a 100 MHz .sup.13 C NMR spectrum of the compound of the
present invention wherein R is a methyl group;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a novel 4-hydroxypyridine derivative
represented by the above-described formula (I), wherein R represents a
methyl group or an isopropyl group. It should be recognized that the
tautomeric isomers of the above-defined compounds of the present invention
fall within the scope of the present invention, which are represented by
the following formula (II):
##STR6##
wherein R represents a methyl group or an isopropyl group.
The compound of the present invention wherein R is an isopropyl group
(hereinafter also referred to as Mer-A2026A) is a yellow oil which is
soluble in commonly available organic solvents and insoluble in water.
This compound has the molecular weight of 4I3 (C.sub.26 H.sub.39 NO.sub.3)
and the following physicochemical properties. The mass spectrum (SIMS)
gives m/z at 414 [(M+H).sup.+ ]. Specific rotation is (solvent: methanol,
c 1.85 at 22.degree. C.):[.alpha.].sub.D =-12.3.degree.. The ultraviolet
absorption spectrum (solvent: methanol) shows maxima at 238.5 nm
(.epsilon.37400) and 223.2 nm(.epsilon.33700). The infra-red absorption
spectrum (solvent: chloroform) gives peaks at 3350, 2950, 2860, 1635,
1590, 1520, 1460, 1390, and 970 cm.sup.-1. The 400 MHz proton magnetic
resonance spectrum as shown in FIG. 1 of the accompanying drawing shows
the following signals (.delta.ppm; in CDCl.sub.3 ; TMS as internal
standard): 0.82(3H,d,J=6.6Hz), 0.94(3H,d,J=6.6Hz), 0.96(3H,d,J=6.6Hz),
1.64(3H,s), 1.71(3H,s), 1.79 (3H,s), 2.03(3H,s), 2.55(1H,m), 2.67(1H,m),
2.81(2H,d,J=7.0Hz), 3.36(2H,d,J=7.0Hz), 3.61(1H,d,J=8.8Hz), 3.78(3H,s),
5.21(1H,d,J=9.5Hz), 5.24(1H,d,J=10.6Hz), 5.30(1H,brt,J=6.6Hz),
5.57(1H,td,J=7.0 and 15.4Hz), 5.96(1H,brs), and 6.09(1H,d,J=15.4Hz). The
100 MHz carbon magnetic resonance spectrum as shown in FIG. 2 of the
accompanying drawing shows the following signals (.delta.ppm; in
CDCl.sub.3 ; the signal of the solvent at 77.0 ppm from TMS is used as a
reference): 10.1(q), 10.8(q), 13.1(q), 16.5(q), 17.4(q), 22.7(q), 22.8(q),
26.8(d), 32.1(t), 36.9(d), 43.0(t), 54.6(q), 82.7(d), 92.4(d), 115.8(s),
120.1(d), 125.8(d), 132.4(s), 133.5(d), 135.6(s), 136.2(d), 136.9(d),
138.0(s), and 160.4(s).
The compound of the present invention wherein R is a methyl group
(hereinafter in the specification, this compound can also be referred to
as Mer-A2026B) is a yellow oil which is soluble in commonly available
organic solvents and insoluble in water. This compound has the molecular
weight of 385 (C.sub.24 H.sub.35 NO.sub.3) and the following
physicochemical properties. The mass spectrum (FAB) gives positive m/z at
386 [(M+H).sup.+ ] and negative m/z at 384 [(M-H).sup.+ ]. Specific
rotation is (solvent methanol, c 0.36 at 24.degree. C.):[.alpha.].sub.D
=-1.07.degree.. The ultra-violet absorption spectrum (solvent: methanol)
shows the maxima at 238.4 nm (.epsilon.37600) and 223.2
nm(.epsilon.33400). The infra-red absorption spectrum (solvent chloroform)
gives peaks at 3340, 2920, 2860, 1635, 1590, 1520, 1460, 1385, and 970
cm.sup.-1. The 400 MHz proton magnetic resonance spectrum as shown in FIG.
3 of the accompanying drawing shows the following signals (.delta.ppm; in
CDCl.sub.3 ; TMS as internal standard): 0.81(3H,d,J=6.6Hz), 1.63(3H,dd,J=
7.0 and 1.1Hz), 1.64(3H,s), 1.72(3H,d,J=1.1Hz), 1.81(3H,d,J=1.1Hz),
2.03(3H,s), 2.69(1H,m), 2.83(2H,d,J=7.0Hz), 3.36(2H,d,J=7.0Hz),
3.64(1H,d,J=9.2Hz), 3.79(3H,s), 5.24(1H,d,J=9.9Hz), 5.32(1H,brt,J=7.0Hz),
5.49(1H,m), 5.58(1H,td,J=7.0 and 15.4Hz), 5.93(1H,brs), and
6.10(1H,d,J=15.4Hz). The 100 MHz carbon magnetic resonance spectrum as
shown in FIG. 4 of the accompanying drawing shows the following signals
(.delta.ppm; in CDCl.sub.3 ; the signal of the solvent at 77.0 ppm from
TMS is used as a reference): 10.1(q), 10.6(q), 13.1(q), 16.6(q), 17.4(q),
36.9(d), 43.0(t), 54.6(q), 82.8(d), 92.4(d), 123.5(d), 125.8(d), 133.6(d),
135.6(s), 135.7(s), and 136.3(d).
In accordance with another embodiment of the present invention, there is
provided a method for preparing Mer-A2026 selected from the group of
Mer-A2026A and Mer-A2026B which comprises the steps of cultivating a
Mer-A2026-producing strain belonging to Streptomyces karnatakensis in an
aqueous nutrient medium, and recovering the Mer-A2026 from the culture.
An example of the Mer-A2026-producing microorganisms useful for the method
for preparing Mer-A2026 is a newly discovered species of actinomycetes
isolated from a soil sample collected in the riverside of Hikichi river,
Fujisawa city, Kanagawa, Japan in 1989 (hereinafter referred to as
Streptomyces karnatakensis Me2108).
Streptomyces karnatakensis Me2108 shows the following morphological,
cultural and physiological characteristics:
1. Morphological characteristics
The aerial hyphae of the cultured strain is well branched and the top of
the aerial hyphae is closed spiral. After growing up, it becomes divided
and forms a spiral chain of conidia. The size of spherical or elliptical
conidium is about 1.0.times.1.0-1.2 .mu.m. The surface of the conidium is
hairy with a lot of spines. No flagellum is observed.
2. Cultural characteristics
The strain has the following cultural characteristics when grown On media
as described below at 30.degree. C. The color of the surface of colony is
indicated according to the symbols described in color Harmony Manual.
(i) Yeast malt agar: Good growth with a lot of epiphytal aerial hyphae and
conidium. The color of the surface of colony is faint gray reddish brown
(5fe). Melanoid pigment and soluble pigment are not observed.
(ii) Tripton yeast agar: Moderate growth with slight aerial hyphae
formation. The color of the surface of colony is white (a). Very small
amount of or almost no epiphytal conidium is observed. Melanoid pigment
and soluble pigment are not observed.
3. Carbon source utilization
The carbon source utilizing pattern according to PridhamGottlieb medium are
summarized in the following Table 1:
TABLE 1
______________________________________
Source of carbon
Growth
______________________________________
L-Arabinose -
D-Xylose -
D-Glucose +
D-Fructose -
Sucrose -
Inositol -
L-Rhamnose -
Raffinose -
D-Mannitol -
______________________________________
-: Not utilized
+: Utilized
4. Physiological characteristics
L,L-Diaminopimelic acid is observed as one of the components of cell wall
when cell wall is analyzed by cellulose thin layer chromatography after
hydrolyzation. No particular pattern of sugar components is observed. The
type of cell wall is classified as Type I.
From the foregoing bacterial characteristics, it is apparent that the
bacterial strain Me2108 belongs to Streptomyces. Further, these bacterial
characteristics are identical with those of Streptomyces karnatakensis
published and authorized by International Streptomyces Project (ISP),
except that the utilization of fructose is described as being.+-.in the
ISP publication. The inventors of the present invention thus concluded
that the bacterial strain Me2108 belongs to Streptomyces karnatakensis.
This strain has been deposited at Fermentation Research Institute, Agency
of Industrial Science and Technology, 1-1-3, Higashi, Tsukuba, Ibaraki
305, Japan, under the access number FERM BP-3583. The microorganism has
been deposited under the Budapest Treaty and all restrictions to access
will be removed upon the grant of a patent.
It is to be understood that, for the preparation of MerA2026, the present
invention is not limited to the use of the particular organism described
herein, which is given for illustrative purpose. This invention also may
include the use of mutants produced from the described organism by
conventional means, such as X-rays, ultra-violet radiation, nitrogen
mustard, or the like.
The Mer-A2026 can be prepared by cultivating a strain which belongs to
Streptomyces karnatakensis and produces Mer-A2026 in a medium containing
assimilable nutrients such as the sources of carbon and nitrogen, and
inorganic salts. Any known nutrients used for the cultivation of
actinomyces may be utilized in the medium.
Examples of the source of carbon in the medium include, for example,
glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses,
animal oil, or vegetable oil. Examples of the source of nitrogen atom
include, for example, soybean meal, wheatgerm meal, corn steep liquor,
cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate,
sodium nitrate, and urea. These carbon and nitrogen sources may preferably
be used in combination. If desired, mineral salts may be added to the
medium. Examples of the salts include, for example, sodium salts,
potassium salts, calcium salts, magnesium salts, cobalt salts, chlorides,
phosphates, or sulfates. Furthermore, appropriate organic or inorganic
materials, which enhance the growth of bacteria and the production of
Mer-A2026 in the medium, may be added to the medium.
For the production of Mer-A2026, aerobic cultivation, preferably, submerged
aerobic cultivation may be used. The fermentation may be conducted at a
temperature of from 15.degree. to 30.degree. C., generally at 28.degree.
C. The productivity of Mer-A2026 generally depends on the cultural medium
and the conditions of cultivation, however, the cumulative amount of
Mer-A2026 will generally reach a maximum in about two to ten days where
shaking culture or tank cultivation is carried out. The 4-hydroxypyridine
derivative Mer-A2026A and MerA2026B can be recovered from the culture
medium and purified after the maximum amount of Mer-A2026A and Mer-A2026B
is cumulated.
The recovery and purification of the 4-hydroxypyridine derivative
Mer-A2026A and Mer-A2026B can be conducted by conventional means utilizing
the physicochemical properties of these compounds. Such separation methods
include, for example, extraction with solvent, adsorption column
chromatography, gel filtration, precipitation, and combinations thereof.
For example, the 4-hydroxypyridine derivative of the present invention,
accumulated mostly in the cultured broth, can be extracted in an organic
layer using a water-immiscible organic solvent such as butanol. Partial
purification can be conducted by the steps of, for example, evaporating
the solvent, dissolving the residue in an organic solvent, removing
impurities as precipitates by adding ether to the solution, filtering the
solution to obtain filtrate containing Mer-A2026, and evaporating the
solvent to afford a dark brown oily residue containing Mer-A2026A and/or
Mer-A2026B.
Further purification of Mer-A2026A and/or Mer-A2026B can be conducted by
applying the resulting residue on a chromatography column using an
adsorbent such as, for example, Sephadex LH-20 (Pharmacia) or silica gel
(such as Wakogel C-200 available from Wako Pure Chemicals, or Kieselgel 60
available from Merck).
In accordance with a further embodiment of the present invention, there is
provided a pharmaceutical composition comprising an effective amount of
the 4-hydroxypyridine derivative of the present invention together with a
pharmaceutically-acceptable carrier or coating. The pharmaceutical
composition is useful as a vasodilator and vasodepressor for the treatment
of circulatory diseases including diseases of cardiovascular system. The
following experimental results clearly demonstrate these pharmacological
activities of the compounds of the present invention.
Vasodilating activity
Male Sprague-Dawley rats weighing 250-300 g were sacrificed by cervical
dislocation and exsanguinated. The thoracic aorta was dissected out and
helical strips, 2 mm wide and 10 mm long, were prepared. Each preparation
was mounted in a 10 ml organ bath containing physiological saline solution
(Krebs-Henzeleit solution, pH 7.4). The solution was gassed with 95%
O.sub.2 - 5% CO.sub.2 at 37.degree. C. and a resting tension of 0.5 g was
applied. Tissues were allowed to equilibrate for at least 60 minutes
before the application of test substances. The developed tension was
measured isometrically with a force-displacement transducer and recorded
on pen-recorder. Tissues had been contracted with 10.sup.-7 M
norepinephrine or isotonic 60 mM KCl and test substances were applied
cumulatively.
As shown in Table 2, the potency of each test substance for the relaxation
was expressed as IC.sub.50 (concentration needed to induce a 50% decrease
in norepinephrine - or KCl-induced contraction). Both Mer-A2026A and
Mer-A2026B more greatly relaxed the aorta than papaverine which commonly
used as a vasodilator.
TABLE 2
______________________________________
IC.sub.50 (M)
Compound Norepinephrine
Isotonic KCl
______________________________________
Mer-A2026A 2.0 .times. 10.sup.-7
1.0 .times. 10.sup.-6
Mer-A2026B 7.7 .times. 10.sup.-7
4.0 .times. 10.sup.-7
Papaverine 4.7 .times. 10.sup.-6
1.7 .times. 10.sup.-5
______________________________________
Depressor effect
Male spontaneously hypertensive rats (SHR rats), 25-31 weeks old, were
anesthetized with 40 mg/kg of pentobarbital sodium injected
intraperitoneally. For the measurement of blood pressure, a cannula was
inserted into the left carotid artery and connected to a multipurpose
polygraph through a pressure transducer. Each animal was allowed to
equilibrate for at least 30 minutes before injection and test substances
were injected into the femoral vein through a polyethylene tube.
As shown in Table 3, Mer-A2026A markedly prolonged the duration time and
decreased the blood pressure. These results indicate that Mer-A2026A is an
effective vasodepressor.
TABLE 3
______________________________________
Compound Dose (.mu.g/kg)
Vasodepression
Duration time
______________________________________
Mer-A2026A
10 3.6 0.2
30 25.0 9.4
100 47.7 24.5
Papaverine
1000 52.0 2.2
______________________________________
Toxicity
The acute toxicity of Mer-A2026A was determined in ICR male mice. Five mice
were used at each dose. An intravenous injection of 500 .mu.g/Kg
sacrificed the one mouse, but at 250 .mu.g/Kg all survived.
The compound of the present invention may be administered to a patient as a
pharmaceutical composition such as a composition for oral administration,
parenteral administration, or topical administration. The pharmaceutical
composition may also be in the form of bathing agent. The pharmaceutical
composition comprises an effective amount of the compound of the present
invention together with a pharmaceutically-acceptable carrier or coating.
The pharmaceutical composition is useful as vasodilator and vasodepressor
for the treatment of circulatory diseases.
The pharmaceutical composition suitable for oral administration may be in a
form of, for example, tablets, capsules, granules, or powder, which can be
prepared by mixing 0.1 to 500 mg of the compound of the present invention
with carriers such as, for example, lactose or starch, and formulating the
mixture, if desired. The pharmaceutical composition suitable for oral
administration may also be in a form of solution, syrup, or ampuled liquid
medicine, which can be prepared by dissolving or suspending 0.1 to 500 mg
of the compound of the present invention in an aqueous medium such as, for
example, purified water or distilled water. The pharmaceutical composition
suitable for parenteral administration may be an injection, which can be
prepared by dissolving or suspending 0.1 to 20 mg of the compound of the
present invention in an aqueous medium such as, for example, distilled
water for injection. The pharmaceutical composition suitable for topical
application may be a hair tonic or a hair grower which can be prepared as
a tonic by using a solvent such as ethanol. The pharmaceutical composition
suitable for topical application may also be a toiletry such as a bathing
agent for comfortable bathing.
The dose of the pharmaceutical composition of the present invention for an
adult patient may genarally be from about 10 to 30 mg per day for oral
administration, which may be increased or decreased depending on the
conditions of the patient to be treated.
The present invention will be further illustrated by the following Examples
and Reference Examples. The Examples are given by way of illustration only
and are not to be construed as limiting. It will be recognized by one
ordinary skill in the art that the compound of the present invention
described above can be prepared by various methods which may include any
one of the steps of producing said compound by cultivating a microorganism
in a medium, concentrating the culture, extracting the substances, and
purifying said compounds. In Examples, all percentages are based on
percents by weight unless otherwise specifically mentioned.
EXAMPLE
The inoculum medium had the following ingredients: 2.0% glycerol; 2.0%
glucose; 2.0% soybean meal; 0.5% yeast extract; 0.25% sodium chloride;
0.32% calcium carbonate; and 0.2% of metal salt solution containing 0.25%
copper sulfate, 0.25% manganese chloride, and 0.25% zinc sulfate. The
production medium was the same as the inoculum medium except that 2.0%
potato starch was used instead of 2.0% glycerol. An antifoaming agent
(0.05%) was added to the culture medium for jar fermentation. The medium
was adjusted to pH 7.4 before being sterilized.
The inoculum medium (50 ml) in a 500 ml Erlenmeyer flask was sterilized at
120.degree. C. for 15 minutes. The medium was then inoculated with a
loopful of the slant culture of Streptomyces karnatakensis Me2108. A
shaking cultivation was carried out for three days at 28.degree. C. to
prepare a first-stage inoculum. The production medium (5 liter) was put
into each three 10 liter jar fermentors and sterilized for 30 minutes at
120.degree. C. To the production medium, each 100 ml of the first-stage
inoculum was added and the medium was cultured with stirring (300 rpm) and
aeration (1 vvm) at 28.degree. C. for 4 days.
After the fermentation, the supernatant collected by centrifugation was
extracted with 12 liter of butanol and the solvent was evaporated under a
reduced pressure to give a residual dark-brown oil. The oil was dissolved
in 200 ml of methanol, and then 800 ml of ether was added to precipitate
impurities which were then removed by filtration. The filtrate was
concentrated and the resulting residue was then dissolved in a small
amount of methanol. The methanol solution was applied on a column of
Sephadex LH-20 (400 ml), and fractions containing active substances were
collected using methanol as an eluent. The fractions were concentrated to
give residue containing active substances.
The residue was dissolved in chloroform-methanol (50:1), and the solution
was applied to a column of 250 ml silica gel (Wakogel C-200). Fractions
were obtained by using each 800 ml of chloroformmethanol (50:1),
chloroform-methanol (20:1), and then chloroformmethanol (10:1) as eluents.
The fractions eluted in chloroformmethanol (20:1) were collected and the
solvent was evaporated. The resulting residue was then dissolved in a
small volume of tolueneacetone (5:1) and the solution was applied on a
column of 50 ml silica gel (Kieselgel 60, Merck). Fractions were obtained
using each 150 ml of toluene-acetone (5:1), toluene-acetone (2:1), and
then toluene-acetone (1:1) as eluents.
The fractions eluted in toluene-acetone (2:1) were collected and the
resulting residue was applied on a preparative thin-layer chromatography
(Kieselgel 60 F.sub.254, Merck) using toluene-acetone (1:1) as a
developer. Fractions at Rfs 0.53 and 0.51 having ultraviolet absorption
were separately collected as Fraction A and Fraction B, respectively.
Fraction B was again chromatographed under the same conditions described
above for further purification. Fraction A and Fraction B were applied to
a gel filtration using Sephadex LH-20 (100 ml) to afford Mer-A2026A (53.5
mg) and Mer-A2026B (9.9 mg), respectively.
One of ordinary skill in the art will recognize that improvements and
modifications may be made while remaining within the scope and spirit of
the present invention. The scope of the present invention is determined
solely by the appended claims.
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